Abstract. A true enigma of modern medicine has persisted for over 150 years. The mechanism by which volatile anesthetics (VAs) produce reversible loss of consciousness and render an organism insensate to pain remains an unsolved mystery. We demonstrated that mitochondrial complex I, an entry point and rate limiting component of the mitochondrial electron transport chain, specifically controls the sensitivity of C. elegans to volatile anesthetics. We also found that complex I function controls anesthetic sensitivity in humans and in mice. Since complex I dysfunction causes VA hypersensitivity in organisms from nematodes to man, the implication is that an ancient mechanism is at hand, linking mitochondrial function to synaptic silencing in the presence of VAs. We extended our molecular studies into mice using the knockout animal, Ndufs4. The Ndufs4(KO) mice lack a subunit of mitochondrial complex I, which results in metabolic dysfunction. We anesthetized young Ndusf4 and wild-type (WT) mice with isoflurane or halothane. For either VA, the KO mice became unresponsive to a tail pinch at a dose ~3-fold lower than for controls. Ndufs4(KO) was also hypersensitive to VAs using loss of righting reflex as the endpoint. These KO mice display the greatest change in VA sensitivity described in a mammal. Testing cell-specific Ndufs4(KO) mice, we found that VA sensitivity was fully controlled by glutamatergic expression of the mutation, with no effect from GABAergic, cholinergic or glial cell expression. We also measured the EC50s for loss of righting reflex (LORR) of the KO mice for other anesthetics whose targets are well characterized. Surprisingly, the animals were actually resistant to the effects of ketamine. The effects of the Ndufs4(KO) are specific in terms of anesthetic and not simply the result of generalized CNS depression. However, the question remains, how do complex I defects affect VA sensitivity. Electrophysiologic studies showed that excitatory synaptic frequencies were decreased by isoflurane in Ndufs4(KO) hippocampal slices. Under conditions of high energetic demand, the function of Ndufs4(KO) excitatory synapses is depressed compared to WT and highly sensitive to isoflurane in a pattern that suggests defective neurotransmitter recycling. Neurotransmitter endocytosis has been shown by others to be dependent on mitochondrial ATP production. We hypothesize that the VA hypersensitivity in Ndufs4 and control mice is mediated through a mitochondrial effect on endocytosis in excitatory neurons. Our aims are to characterize the mechanism underlying the defective endocytosis of neurotransmitter. Our overarching goal is to understand how the VAs function. We have linked mitochondrial function to behavior in VAs in worms, mice, and man. Our proposed studies are aimed to identify the basic molecular mechanisms of action of VAs.